Abstract: Microglia are key players in most if not all central nervous system (CNS) disorders including neuropathic pain, Rett syndrome, amyotrophic lateral sclerosis and Nasu-Hakola disease. Recent evidence suggests that these cells also participate in regulation of neurogenesis, circuit remodeling and synaptic plasticity during normal postnatal development, influences they may continue to exert in the adult brain. Despite these vital roles in normal physiology and pathology, we know virtually nothing about these cells' signaling, how it is influenced by local environment and disease, and how it influences other cells and their dynamics in the normal brain. This dearth of knowledge is largely due the inability of in vitro approaches to accurately recapitulate the brain's environment to which microglia are highly sensitive and adaptable and a lack of proper tools to monitor and manipulate microglial cells in vivo. To begin to overcome these limitations we have developed a set of fluorescence staining and genetic manipulation, imaging and data analysis tools for the study of cellular dynamics in the normal brain. These tools have allowed us to reveal, for example, novel forms of astrocytic and neuronal excitation in behaving mice and microglia's surveillance behavior in the healthy adult brain. Building on these approaches we now propose to develop a new set of tools for minimally invasive monitoring and manipulation of microglia in both superficial and deep brain structures. This will allow us to deliver unprecedented insight into these cell's functional dynamics and interactions. Our research therefore has broad implications for our view of microglia, their beneficial and detrimental roles in the healthy and diseased brain and more generally for our ability to uncover complex cell-cell interactions in the normal brain. Public Health Relevance: Microglial cells are involved in onset, progression and/or resolution of essentially all brain pathologies. However, little is known about their properties in the intact brain. We propose to fill this essential gap in our knowledge through development and application of novel research tools.